Short Communications

Supercritical Fluid Extraction of rigida Adnan O¨ zcan Anadolu University, Faculty of Science, Department of Chemistry, 26470 Eskisehir, Turkey

Asiye Safa O¨ zcan* Balikesir University, Faculty of Art and Science, Department of Chemistry, 10100 Balikesir, Turkey

Ms received: July 27, 1999; accepted: February 29, 2000 Key Words: Supercritical fluid extraction; capillary GC; hydrocarbons; bio-oil

1 Introduction In this study, supercritical CO2 was used to extract aliphatic The steadily increasing consumption of crude oil and appar- hydrocarbons with recoveries comparable to those of other ent insecurity in the supply and price of foreign petroleum conventional extraction techniques [13–15]. After SFE of during the late 1970’s have boosted research on the substitu- Euphorbia rigida, quantitative analysis of the hydrocarbons tion of traditional hydrocarbon fuels by alternative renewable was performed by gas chromatography. Finally, GC/MS ana- resources for feedstock chemicals [1]. lysis was undertaken to confirm the identity of the hydrocar- bons and some of the polar compounds such as aldehydes, Euphorbia rigida, a member of the family, is alcohols, esters, etc. one such renewable source. Not suited for food production it grows abundantly in arid and semi-arid regions in western 2 Experimental and south-western Anatolia, Turkey [2–4]. This family of includes roughly 2000 , ranging from small 2.1 Sample herbs to large trees. The majority of them can produce a Euphorbia rigida was collected from south-western Anatolia milky latex that yields wide range of chemical such as rubber, between Afyon and Denizli. The plants were harvested oils, terpenes, waxes, hydrocarbons, starch, resins, tannins, between April to June, dried, and stored in a cool and a dark and balsams of interest to various industries. In recent years room for six months. The plants’ leaves and stalks were Euphorbia species have become attractive as petro-crops ground in a blender to produce a fine powder. because of their hydrocarbon potential. Several species of Euphorbia such as Euphorbia lathyris and Euphorbia tiru- calli are being cultivated to evaluate their potential as bio- 2.2 Extraction Procedure sources of chemical feedstock [2, 5, 6]. Supercritical fluid extractions were performed using SFE grade CO2 and an Isco Model 100 D syringe pump operated Soxhlet extraction and pyrolytic conversion are the tradi- at 400 atm; CO2 was cooled to –108C and –58C (Julabo F10 tional methods for extraction of bio-oil and natural products cooler). The material was mixed with (1:1) glass beads from the plant material, but the former is labor intensive and (Alltech Associates, 100 lm o.d. silanized) prior to loading time consuming, requires large volumes of solvents, and pol- into an extraction cell (2.2 mL volume cell from Keystone lutes the environment [7–9]. Pyrolytic techniques are equally Scientific). The cell was placed in an extractor (Isco SFX- undesirable due to the high temperatures used, also entailing 2.10) which consists of a temperature controller, a vent valve, loss of essential oils. With the demand for more environmen- an on/off valve, an extraction cell, and another on/off valve tally friendly methods and increased productivity, supercriti- to maintain the extraction cell at the required temperature. cal fluid extraction (SFE) has been evaluated. This method The extractor was connected with the restrictor via a finger- offers several advantages, such as low viscosities, high diffu- tight union (Keystone). The flow rate of the supercritical sivities and fast mass transfer, leading to rapid extraction. fluid through the extraction cell was measured as liquid CO2 Although many supercritical fluids have been used in SFE, at the pump and was controlled by 10 cm long restrictors the most popular one is CO2 because of its easily attainable (30 lm i.d.) cut from fused-silica tubing. Extracted analytes 8 supercritical condition at 75.3 atm and 31 C. Carbon dioxide were collected in a 21 mL collection vial with a screw cap is also readily available in a highly pure state. It is inexpen- (with a hole) and PTFE-laminated silicon septa. Methylene sive, non-polar, non-toxic, chemically inert, and is able to chloride (5 mL) was used as a trapping solvent. solvate a wide range of organic compounds including those having higher molecular mass. However, the limitation of The plant material was sequentially extracted with pure CO2 CO2 is that the polar organic compounds are often difficult to (at 400 atm, 508C) for 30 min, followed by CO2 + 10% extract from plant materials though they are soluble in super- CH3OH (v/v) (at 400 atm, 508C) first in the static mode for critical CO2. The extraction of polar molecules requires addi- 15 min to accomplish equilibrium in the cell and subsequently tion of a modifier, most commonly methanol [10–13]. in the dynamic mode for 30 min. Fractions were collected at

J. High Resol. Chromatogr. 2000, 23, (5) 397–400 i WILEY-VCH Verlag GmbH, D-69451 Weinheim 2000 0935-6304/2000/0505–0397$17.50+.50/0 397 SFE of Euphorbia rigida

set time intervals for both the pure CO2 and CH3OH-modified Table 1. The percentages of extract by SFE and Soxhlet and the per- CO2 extractions. The accuracy of the temperature and the pres- centages of hydrocarbons in SFE and Soxhlet. sure measurements (of the full scale of the pump) were l18C and l2%, respectively. Measurements were carried out in tri- Extraction Conditions Extractable plant Extractable hydrocar- plicate with a standard deviation of less than 0.6%. Type material (wt %) bons from plant (wt %)

a) a) After SFE, the sample (residue) was removed from the SFE CO2/50 8C 4.02 l 0.60 0.21 l 0.04 l a) l a) extraction cell and placed in a vial and sonicated with 10 mL CO2 + 10% 1.99 0.13 0.06 0.003 8 of CH2Cl2 for 4 h. The solvent was then evaporated to 1.5 mL CH3OH/50 C Residueb) 1.48 l 0.22a) – and C19-nonadecane was added as internal standard to the l a) l a) sample for GC-FID analysis. The SFE recoveries from Soxhlet 8 h/CH2Cl2 8.64 0.039 0.19 0.02 Euphorbia rigida were then compared with the hydrocarbon a) Values in l are the standard deviation of triplicate extractions. recoveries obtained by the Soxhlet process. b) Sonication of plant residue in CH2Cl2 for 4 h. To determine 100% recovery, the plant material (1 g) was (0.19%) was lower than by SFE (0.21%). The yield of hydro- placed in a cellulose thimble, transferred to a Soxhlet extrac- carbons after column fractionation from SFE was 0.27 wt %, tor and extracted for 8 h with 75 mL CH2Cl2. A vacuum eva- i.e. 30% more hydrocarbons than the Soxhlet (0.19%) porator was then used for evaporation of the solvent at 308C. extract. Thus, supercritical CO2 extracted the majority of the The SFE and Soxhlet extracts were fractionated in a silica- hydrocarbons. The modifier was used to recover more polar gel column with pentane to recover the hydrocarbon fraction. high molecular weight hydrocarbons. First, silica-gel (Fisons, 30–70 mesh) was dried at 1708C and then placed in a chromatographic column (45 cm61.6 Not all of the extractable material consisted of hydrocarbons; cm i.d.). Samples were loaded onto the column and eluted some pigments such as taraxanthin (413 nm), lycopene with 75 mL pentane. Fractions were collected in a 100 mL (456 nm), and chlorophyll (a) (665 nm) are also extracted, as flask and pentane was evaporated off at 308C under vacuum. determined by UV spectroscopy [16].

UV spectra (Unicam UV2-100) were recorded in CH3OH by 3.2 GC-FID of SFE and Soxhlet Extracts using 1 cm3 cell. An appropriate wavelength range (190 nm– 900 nm) was chosen to analyze the extracts. The hydrocarbons pre-fractionated with pentane using a silica-gel column were determined by GC with the aid of GC-FID analysis was performed (Hewlett-Packard 5890) external and internal standards (Table 2, Figure 1, Figure 2). with helium as carrier gas on a BP1 capillary column 6 l (25 m 0.32 mm i.d.; 0.5 m film thickness) from SGE Table 2. Comparison of CO2, CO2 + 10% CH3OH, sonication in

(Scientific Glass Engineering). The injection port and detec- CH2Cl2, and Soxhlet the recovery of the quantitation of the hydrocarbons tor were both heated at 3108C. The GC oven was ramped from Euphorbia rigida. from 408C (2 min hold) at 15 K min – 1 to 3008C. The split- less injection mode was used. Quantitative determination of Species Concentration (lg g – 1) a) b) the hydrocarbons was based on comparison of peak areas CO2 CO2+10% CH3OH Soxhletc) with those of the internal standard. An n-alkane standard C13 5.8 ND ND from C14 to C40 (Aldrich) (2.91 mg each) was prepared in C15 2.3 ND ND pentane (10 mL) and stored in a refrigerator at 58C. C20 8.7 ND 9.9 GC/MS analysis was carried out on a Carlo Erba model C21 3.9 ND 2.6 HRGC 5160 and a VG-Trio 1000 (Fisons) mass spectrometer. C22 ND ND 11.0 C 16.2 ND 35.9 A BPX5 capillary column (SGE; 25 m60.32 mm i.d.; 23 C ND ND 32.8 0.5 lm film thickness) was used. The mass spectrometer was 24 C25 23.9 4.6 31.9 set to scan between m/z 40 and 400, total ion current (TIC) C26 3.6 ND 7.3 and selective ion monitoring (SIM) modes; the electron- C27 11.7 3.1 24.4 impact ionizing voltage was 70 eV. C28 32.7 ND 6.4

C29 672 10.4 486 3 Results and Discussion C30 2.3 ND ND C31 73.4 8.4 88.1 3.1 Fractionation C33 26.3 2.1 27.1

The yield of Soxhlet extraction of 1 g of Euphorbia rigida See Figure 1 and Figure 2 for chromatographic results. ND = not – 1 – 1 86.4 mg g and that of SFE was 74.9 mg g . These were detected. a) further fractionated using a silica-gel column with pentane as Sample extracted at 400 atm, 508C CO2 for 30 min. b) eluent. As can be seen from Table 1, the percentage of Sample extracted at 400 atm, 508C CO2 + 10% CH3OH for 30 min. c) extractable hydrocarbons obtained by Soxhlet extraction Sample extracted with Soxhlet apparatus, 8 h, in CH2Cl2.

398 VOL. 23, MAY 2000 J. High Resol. Chromatogr. SFE of Euphorbia rigida

Figure 3. Total ion and selected ion GC/MS chromatograms of extract of Euphorbia rigida on a BPX5 capillary column.

Table 3. The identification of some tetracyclic triterpenoid compounds by using GC/MS (Numbers refer to Figure 3). Figure 1. GC analysis of SFE sample from Euphorbia rigida on a BP1 capillary column. A) 508C, 400 atm CO2 B) 508C, 400 atm CO2 + 10% Numbers Name of compounds CH3OH C) Sonication in CH2Cl2. 1 9-Octadecenal 2 1-Eicosanol 3 Heneicosyl formate 4 1-Heptacosanol 5 Kauren-18-ol-acetate, (4b) 6 Cholest-8-en-3-ol, 14-methyl-, (3b, 5a)- 7 9,19-Cyclolanostan-3-ol, 24-methylene, (3b)- 8 9,19-Cyclo-9b-lanostane-3b, 25-diol 9 Ergost-25-ene-3,5,6,12-tetrol, (3b, 5a, 6b, 12b) 10 9,19-Cyclolanost-23-ene-3,25-diol-3-acetate, (3b, 23-E)

4 Conclusions SFE has been shown to successfully extract hydrocarbons from Euphorbia rigida. SFE was complete within 60 min, Figure 2. GC analysis of a Soxhlet extract from Euphorbia rigida on a which is eight times faster than Soxhlet extraction. This BP1 capillary column. method was easier to perform and inexpensive; moreover, the consumption of solvent (1–2 mL) was lower than in Soxhlet extraction (75 mL). The recovery of hydrocarbons from the SFE-CO2 extract (0.88 mg g–1) is higher than that of SFE-CO + CH OH modi- 2 3 The per-sample cost of SFE grade CO2 is often only 1 or 2% – 1 – 1 fier (0.029 mg g ) and of Soxhlet extraction (0.76 mg g ). of equivalent extraction solvents. For instance, the SFE extraction of a 1 g sample at an average CO2 flow rate of – 1 3.3 GC/MS of SFE-CO2 Extract 0.21 mL min and 30 min time requires approximately 0.63 g of CO . This cost is less than US Dollar 0.01 as com- The SFE extracts analyzed by GC/MS (Figure 3). Although 2 pared to US Dollar 5–20 for comparable Soxhlet extraction the total ion current chromatogram is quite complex and con- solvents. A significant amount of electric energy can be saved tains several overlapping peaks (including a large hump at as well. The implementation of SFE can eliminate long, high 22–24 min), the reconstructed selected ion current chromato- temperature reflux periods and solvent concentration eva- grams for alkanes (m/e = 57) and alkenes (m/e = 55) show poration steps. Furthermore, laboratory venting costs can be clearly resolved chromatographic peaks that could be used reduced. for quantification of the individual species [17]. This extract consists of some alkanes, alkenes, and a free fatty acid, alco- Since supercritical fluids possesses low viscosity, high diffu- hols, an ester, an aldehyde, and some tetracyclic triterpenoid sivity and hence fast mass transfer is achieved, this leads to compounds (Table 3). rapid extraction than Soxhlet extraction. The use of CO2 as a

J. High Resol. Chromatogr. VOL. 23, MAY 2000 399 SFE of Euphorbia rigida supercritical extraction fluid also reduces hazards to the [6] M. Calvin, Pure Appl. Chem. 1978, 50, 407. environment. [7] E. Bondar, M. Koel, M. Liiv, Fuel 1998, 77, 215. [8] M.C. Lin, M.J. Tsai, K.C. Wen, J. Chromatogr. A 1999, 830, 387. Acknowledgment [9] C.T. da Costa, S.A. Margolis, B.A. Benner Jr., D. Horton, J. Chro- The authors acknowledge Prof. Dr. K.D. Bartle and Prof. Dr. A.A. Clif- matogr. A 1999, 831, 167. ford at the School of Chemistry, University of Leeds, for providing the [10] J.W. Hills, H.H. Hill, J. Chromatogr. Sci. 1993, 31, 6. apparatus used in this work. [11] M.D. Burford, S.B. Hawthorne, D.J. Miller, Anal. Chem. 1993, 65, 1497. References [12] N.J. Cotton, K.D. Bartle, A.A. Clifford, C.J. Dowle, J. Appl. Poly- [1] J.B.D. Sheldon, in: Biomass for Energy, Industry and Environment, mer Sci. 1993, 48, 1607. 6th E. C. Conference, G. Grassi. A. Collina, H. Zibetta (eds), Else- vier Applied Science, London-New York 1992, p. 1067. [13] M.D. Burford, S.B. Hawthorne, D.J. Miller, J. Chromatogr. A 1994, 685, 95. [2] K. Seshagirifao and M.N.V. Prasad, in: Biomass for Energy, Indus- try and Environment, 6th E. C. Conference, G. Grassi. A. Collina, [14] V. Lopez-Avila, J. Benedicto, N.S. Dodhiwala, R. Young, W.F. H. Zibetta (eds), Elsevier Applied Science, London-New York Beckert, J. Chromatogr. Sci. 1992, 30, 335. 1992, p. 1342. [15] S.E. Eckert-Tilotta, S.B. Hawthorne, D.J. Miller, Fuel 1993, 72, [3] M. Calvin, Chem. Eng. News 1978, 56, 30. 1015. [4] M. Calvin, Science 1983, 219, 24. [16] DMS UV Atlas of Organic Compounds, Butterworth, London 1971, Chap. 5. [5] E.K. Nemethy, J.W. Otvos, M. Calvin, Pure Appl. Chem. 1981, 53, 1101. [17] S.B. Hawthorne, D.J. Miller, J. Chromatogr. Sci. 1986, 24, 258.

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